Appendix B. Chemical analysis.
The dried and ground V. hirundinaria leaves (20 mg material per sample) were extracted twice (2×1 h) with acetone/water (ratio 7/3, 2×500 µL) and once (1×1 h) with dichloromethane/methanol (ratio 1/1, 1×500 µL) on a planary shaker. Supernatant solutions were separated after each extraction step by centrifugation in an Eppendorf centrifuge (10 min, 14000 rpm). Pooled supernatant solutions were concentrated into aqueous phase by an Eppendorf concentrator, the residues frozen and freeze-dried. 500 µL water and 1000 µL hexane were added into Eppendorf tubes containing the lyophilized residues. Tubes were shaken for 10 min to let the water-soluble compounds dissolve into water and the lipophilic compounds into hexane. The separation of the two phases was enhanced by centrifugation (10 min, 14000 rpm). 800 µL of the hexane-phase was pipetted into a separate eppendorf tube and evaporated into dryness by an Eppendorf concentrator. The residue was dissolved into 500 µL ethanol; this solution was used for the analysis of lipophilic compounds in leaves of V. hirundinaria. The concentration of water-soluble compounds was analysed directly from the water-phase.
HPLC-DAD analysis of leaf extracts was performed at 200 nm, 256 nm, 315 nm, 280 nm, and 349 nm with Merck-Hitachi’s LaChrom HPLC system (Merck-Hitachi, Tokyo, Japan). To maximize the quality of the chemical analyses with a Merck Chromolith Performance RP-18e (100 × 4.6 mm) column, separate HPLC methods were developed for both lipophilic and water-soluble compounds of V. hirundinaria. Two solvents were used in both methods: (A) 0.05 M phosphoric acid and (B) acetonitrile. Injection volume was 10 µL and the acquisition of UV-spectra was done at 195450 nm. Water-soluble compounds were quantified using gallic acid, chlorogenic acid, and an isolated flavonoid fraction of V. hirundinaria as external standards. Lipophilic compounds were quantified using an isolated lipophilic fraction of V. hirundinaria as an external standard.
Selected extracts and chemical fractions of V. hirundinaria leaves were analysed also with HPLC-ESI-MS using a Perkin-Elmer Sciex API 365 triple quadrupole mass spectrometer (Sciex, Toronto, Canada) equipped with an ion-spray interface. The HPLC system consisted of two Perkin-Elmer Series 200 micro pumps (Perkin-Elmer, Norwalk, CT, USA) connected to a Series 200 autosampler. The column used, and chromatographic and ESI-MS conditions were as described previously (Salminen et al. 1999). Compounds were characterized on the basis of their UV and mass spectra, and mass spectral fragmentation.
Salminen, J.-P., V. Ossipov, J. Loponen, E. Haukioja, and K. Pihlaja. 1999. Characterisation of hydrolysable tannins from leaves of Betula pubescens by high-performance liquid chromatography mass spectrometry. Journal of Chromatography A 864:283291.